Many diseases and especially cancers are associated with genetic mutations such as single point mutations, small base pair insertions/deletions and the like. Almost all current methods for detection of these rare alleles rely on polymerase chain reaction (PCR). However, a major limitation of PCR-based methods is their low sensitivity and preferential amplification of normal (wild-type) sequence due to their greater relative abundance within a sample. Often, detection of a mutant allele is not possible until it represents greater than 5-10% of the total alleles present. Thus, the ability to detect genetic mutations in a background of wild-type DNA sequence where the variant sequence is present at a low percentage relative to non-variant (target) sequence is a beneficial and highly desired.
Modified PCR methods allowing selective amplification of mutant genes without requiring post-amplification sequencing assays have been described. Such methods include Restriction endonuclease-mediated selective PCR: a novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol 153:373-379), Detection of tumor mutations in the presence of excess amounts of normal DNA. Nat Biotechnol 20:186-189; “locked nucleic acid”) COLD-PCR (co-amplification at lower denaturation temperature PCR) (Li, J., et al., 2008. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579-584.), US20130149695 (Method for detecting genetic mutation by using a blocking primer), U.S. Pat. No. 8,623,603 (Full cold-PCR enrichment with reference blocking sequence); U.S. Pat. No. 8,455,190 (Enrichment of a target sequence); WO2003072809 (Melting Temperature Dependent DNA Amplification) and others.
COLD-PCR technique is relatively simple to perform, but has a low amplification factor (3-100×) and a low sensitivity towards minute temperature changes (Li, J., et al., 2008. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579-584, Luthra, R., et al., 2009. COLD-PCR finds hot application in mutation analysis. Clin Chem 55:2077-2078). Other methods, such as that described in Molloy et al. (WO2003072809) requires use of lower denaturing temperature in order to selectively amplify target sequences. Thus Molloy is applicable only for those target sequences having a lower melting temperature (Tm) than their wild-type sequence.
Nucleic acids in cancerous tissues, circulating cells, and cell-free (cf) nucleic acids present in bodily fluids can aid in identifying and selecting individuals with cancer or other diseases associated with such genetic alterations. Mutations in BRAF and KRAS are examples of genetic alterations that confer a survival and growth advantage to cancer cells. Such genetic alterations can be used for selection of targeted therapies. But in a subject, the alterations are present with a large excess of non-altered, wildtype sequences.
See, e.g., Spindler et al., 2012; Benesova et al., 2013; Dawson et al., 2013; Forshew et al., 2012; Shaw et al., 2012. Some data suggest that the amount of mutant DNA in blood correlates with tumor burden and can be used to identify the emergence of resistant mutations (Forshew et al., 2012; Murtaza et al., 2013; Dawson et al., 2013; Diaz et al., 2012; Misale et al., 2012; Diehl et al., 2008).
There is a need for additional methods whereby a greater sensitivity and/or enrichment of target sequence can be achieved with efficiency and ease. The present invention addresses that need.